Healing of skin defects progresses through three general phases, i.e., (1) inflammation, (2) wound cell migration and mitosis, and (3) extracellular matrix production and remodelling. The ordered sequence of these events is thought to be orchestrated by interactions among cells, growth factors, and extracellular matrix proteins. A crucial step of skin wound healing is epidermal regeneration, i.e., re-epithelialization. Besides interfollicular epidermal keratinocytes from the wound edges, the outer root sheat (ORS) cells from residual hair follicles also contribute to this process (Eisen et al., J Invest Dermatol 15: 145-156, 1955). The ORS of hair follicles is comprised largely of undifferentiated keratinocytes that encompass the cylindrical structures of the hardened inner root sheath and the hair shaft (Montagna and Parakkal, pp. 172-258 in "The Structure and Function of Skin", c. 1974 by Academic Press New York, N.Y.). Recent literature indicates that ORS cells are at a lower level of commitment to differentiation than the basal interfollicular keratinocytes (Coulombe et al., J Cell Biol 109: 2295-2312, 1989; Limat et al., Exp Cell Res 194: 218-227, 1991; Limat et al., Cell Tissue Res 275: 169-176, 1994), and label retaining cells have been detected in the animal as well as the human ORS region near the bulge area which possibly represent stem cells for skin epithelial tissues (Cotsarelis et al., Cell 61: 1329-1337, 1990 Kobayashi et al., Proc Natl Acad Sci USA 90: 7391-7395, 1993; Yang et al., J Invest Dermatol 105: 14-21, 1993; Rochat et al., Cell 76: 1073-1076, 1994; Moll, J Invest Dermatol 105: 14-21, 1995). Human ORS cells isolated from plucked anagen scalp hair follicles can be expanded extensively in vitro (Weterings et al., Brit J Dermatol 104: 1-5, 1981; Limat and Noser, J Invest Dermatol 87: 485-488, 1986; Imcke et al., J Am Acad Dermatol 17: 779-786, 1987; Limat et al., J Invest Dermatol 92: 758-762, 1989). Under conventional submerged culture conditions, ORS cells resemble interfollicular epidermal keratinocytes by both morphologic and biochemical (e.g., keratin profiles) criteria (Stark et al., Differentiation 35: 236-248, 1987; Limat et al., J Invest Dermatol 92: 758-762, 1989; Limat et al., Ann NY Acad Sci 642: 125-147, 1991). In organotypic cocultures with human dermal fibroblasts, i.e., under conditions mimicking the epidermal environment, ORS cells with respect to histological, immunohistological, ultrastructural and biochemical criteria develop a stratified epithelium reminiscent of regenerating epidermis (Lenoir et al., Dev Biol 130: 610-620, 1988; Limat et al., Exp Cell Res 194: 218-227, 1991; Limat et al., Ann NY Acad Sci 642: 125-147, 1991). If such organotypic cultures are grafted onto nude mice, ORS cells form a regular neo-epidermis that is under homeostatic control (Limat et al., Transplantation 59: 1032-1038, 1995). Thus, human ORS cells are of considerable interest for clinical application.
In the last decade, interest has focused on the use of cultured epithelial cells for wound coverage. First, sheets of cultured autologous interfollicular keratinocytes were grafted successfully on acute wounds, mainly in the treatment of larger third degree burns (O'Connor et al., Lancet 1: 75-78, 1981; Compton et al., Lab Invest 60: 600-612, 1989) but also of epidermolysis bullosa (Carter et al., J Am Acad Dermatol 17: 246-250, 1987), pyoderma gangrenosum (Dean et al., Ann Plast Surg 26: 194-195, 1991; Limova and Mauro, J Dermatol Surg Oncol 20: 833-836, 1994), and wounds after excision of giant congenital nevi (Gallico et al., J Plast Reconstr Surg 84: 1-9, 1989) or separation of conjoined twins (Higgins et al, J R Soc Med: 108-109, 1994).
In contrast to the treatment of acute wounds, grafting of chronic wounds such as leg ulcers with cultured keratinocytes has been much less successful. Allografts do not result in a permanent take (Fabre, Immunol Lett 29: 161-166, 1991) and thus may be classified as a quite effective but expensive biological dressing (reviewed by Phillips et al., J Am Acad Dermatol 21: 191-199, 1989). A reproducible, major definite take of autologous keratinocyte grafted by various modalities--sheets of submerged keratinocyte cultures consisting of only a few, noncornified cell layers (Hefton et al., J Am Acad Dermatol 14: 399-405, 1986; Leigh and Purkis, Clin Exp Dermatol 11: 650-652, 1986; Leigh et al., Brit J Dermatol 117: 591-597, 1987; Philips et al., J Am Acad Dermatol 23: 189-198, 1990; Giannotti et al., G Ital Dermatol Venerol 125: 161-167, 1990; Harris et al., Clin Exp Dermatol 18: 417-420, 1993), trypsinized single cells attached to collagen-coated dressings (Brysk et al., J Am Acad Dermatol 25: 238-244, 1991), skin equivalents (Mol et al., J Am Acad Dermatol 24: 77-82, 1991)--has not been convincingly documented. The same holds true for reports on grafting of freshly isolated, autologous interfollicular keratinocytes (Hunyadi et al., J Dermatol Surg Oncol 14: 75-78, 1988) or ORS cells (Moll et al, Hautarzt 46: 548-552, 1995) fixed to the wound bed by a fibrin glue. Disadvantages of bovine serum used during cultivation of the keratinocytes may contribute to reduced take rate, since it resists in keratinocytes (Johnson et al., J Burn Care Rehab 11: 504-509, 1990).